bims-proteo 2024-09-08 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024‒09‒08
forty papers selected by
Eric Chevet, INSERM

The integrated stress response regulates 18S nonfunctional rRNA decay in mammals.
Poly-alanine-tailing is a modifier of neurodegeneration caused by Listerin mutation.
Disordered regions in the IRE1α ER lumenal domain mediate its stress-induced clustering.
Alternating binding and p97-mediated dissociation of SDS22 and I3 recycles active PP1 between holophosphatases.
Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans.
Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway.
The ULK1 effector BAG2 regulates autophagy initiation by modulating AMBRA1 localization.
Multi-protein assemblies orchestrate co-translational enzymatic processing on the human ribosome.
DMDA-PatA mediates RNA sequence-selective translation repression by anchoring eIF4A and DDX3 to GNG motifs.
Metabolically activated proteostasis regulators that protect against erastin-induced ferroptosis.
Misfolding and aggregation in neurodegenerative diseases: protein quality control machinery as potential therapeutic clearance pathways.
The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation.
Decoding the molecular mechanism of selective autophagy of glycogen mediated by autophagy receptor STBD1.
Histone deacetylase inhibition expands cellular proteostasis repertoires to enhance neuronal stress resilience.
Differential effects of 40S ribosome recycling factors on reinitiation at regulatory uORFs in GCN4 mRNA are not dictated by their roles in bulk 40S recycling.
PROTAC-DB 3.0: an updated database of PROTACs with extended pharmacokinetic parameters.
Aggregate-selective removal of pathological tau by clustering-activated degraders.
µMap proximity labeling in living cells reveals stress granule disassembly mechanisms.
Nuclear proteasomes buffer cytoplasmic proteins during autophagy compromise.
Delineation of the substrate recognition domain of MARCHF6 E3 ubiquitin ligase in the Ac/N-degron pathway and its regulatory role in ferroptosis.
ER-associated degradation ligase HRD1 links ER stress to DNA damage repair by modulating the activity of DNA-PKcs.
E3 ubiquitin ligase ANKIB1 attenuates antiviral immune responses by promoting K48-linked polyubiquitination of MAVS.
Mutant induced neurons and humanized mice enable identification of Niemann-Pick C1 proteostatic therapies.
Leveraging multiple data types for improved compound-kinase bioactivity prediction.
Episodic Transport of Protein Aggregates Achieves a Positive Size Selectivity in Aggresome Formation.
Nucleoid-phagy: a novel safeguard against mitochondrial DNA-Induced inflammation.
A p62-dependent rheostat dictates micronuclei catastrophe and chromosome rearrangements.
Disorder-mediated interactions target proteins to specific condensates.
Lipid- and protein-directed photosensitizer proximity labeling captures the cholesterol interactome.
Quantitative mapping of proteasome interactomes and substrates using ProteasomeID.
Proteome and Glycoproteome Analyses Reveal Regulation of Protein Glycosylation Site-Specific Occupancy and Lysosomal Hydrolase Maturation by N-Glycan-Dependent ER-Quality Control.
The stress response regulator HSF1 modulates natural killer cell anti-tumour immunity.
Promotion of endoplasmic reticulum retrotranslocation by overexpression of E3 ubiquitin-protein ligase synoviolin 1 reduces endoplasmic reticulum stress and preserves cone photoreceptors in cyclic nucleotide-gated channel deficiency.
Identification and mechanism of wheat protein disulfide isomerase-promoted gluten network formation.
High-grade serous ovarian cancer development and anti-PD-1 resistance is driven by IRE1α activity in neutrophils.
Regulation of proteostasis by sleep through autophagy in Drosophila models of Alzheimer's disease.
Micronuclear collapse from oxidative damage.
Heat Shock Protein 90 Interactome-Mediated Proteolysis Targeting Chimera (HIM-PROTAC) Degrading Glutathione Peroxidase 4 to Trigger Ferroptosis.
The novel ribosome biogenesis inhibitor usnic acid blocks nucleolar pre-60S maturation.
Engineering artificial non-coding RNAs for targeted protein degradation.

bioRxiv. 2024 Jul 30. pii: 2024.07.30.605914. [Epub ahead of print]

The integrated stress response regulates 18S nonfunctional rRNA decay in mammals.

Aaztli R Coria, Akruti Shah, Mohammad Shafieinouri, Sarah J Taylor, Wilfried Guiblet, Jennifer T Miller, Indra Mani Sharma, Colin Chih-Chien Wu.

18S nonfunctional rRNA decay (NRD) detects and eliminates translationally nonfunctional 18S rRNA. While this process is critical for ribosome quality control, the mechanisms underlying nonfunctional 18S rRNA turnover remain elusive. NRD was originally identified and has exclusively been studied in Saccharomyces cerevisiae. Here, we show that 18S NRD is conserved in mammals. Using genome-wide CRISPR genetic interaction screens, we find that mammalian NRD acts through the integrated stress response (ISR) via GCN2 and ribosomal protein ubiquitination by RNF10. Selective ribosome profiling reveals nonfunctional 18S rRNA induces translational arrest at start sites. Indeed, biochemical analyses demonstrate that ISR activation limits translation initiation and attenuates collisions between scanning 43S preinitiation complexes and nonfunctional 80S ribosomes arrested at start sites. Overall, the ISR promotes nonfunctional 18S rRNA and 40S ribosomal protein turnover by RNF10-mediated ubiquitination. These findings establish a dynamic feedback mechanism by which the GCN2-RNF10 axis surveils ribosome functionality at translation initiation.

DOI: https://doi.org/10.1101/2024.07.30.605914
bioRxiv. 2024 Aug 24. pii: 2024.08.24.608776. [Epub ahead of print]

Poly-alanine-tailing is a modifier of neurodegeneration caused by Listerin mutation.

Hao-Chih Hung, Carlos Costas-Insua, Sarah E Holbrook, Jennifer E Stauffer, Paige B Martin, Tina A Müller, David G Schroeder, Yu Kigoshi-Tansho, Haifei Xu, Rüdiger Rudolf, Gregory A Cox, Claudio A P Joazeiro.

The surveillance of translation is critical for the fitness of organisms from bacteria to humans. Ribosome-associated Quality Control (RQC) is a surveillance mechanism that promotes the elimination of truncated polypeptides, byproducts of ribosome stalling during translation. In canonical mammalian RQC, NEMF binds to the large ribosomal subunit and recruits the E3 ubiquitin ligase Listerin, which marks the nascent-chains for proteasomal degradation. NEMF additionally extends the nascent-chain's C-terminus with poly-alanine ('Ala-tail'), exposing lysines in the ribosomal exit tunnel for ubiquitination. In an alternative, Listerin-independent RQC pathway, released nascent-chains are targeted by Ala-tail-binding E3 ligases. While mutations in Listerin or in NEMF selectively elicit neurodegeneration in mice and humans, the physiological significance of Ala-tailing and its role in disease have remained unknown. Here, we report the analysis of mice in which NEMF's Ala-tailing activity was selectively impaired. Whereas the Nemf homozygous mutation did not affect lifespan and only led to mild motor defects, genetic interaction analyses uncovered its synthetic lethal phenotype when combined with the lister neurodegeneration-causing mutation. Conversely, the lister phenotype was markedly improved when Ala-tailing capacity was partially reduced by a heterozygous Nemf mutation. Providing a plausible mechanism for this striking switch from early neuroprotection to subsequent neurotoxicity, we found that RQC substrates that evade degradation form amyloid-like aggregates in an Ala-tail dependent fashion. These findings uncover a critical role for Ala-tailing in mammalian proteostasis, and deepen our molecular understanding of pathophysiological roles of RQC in neurodegeneration.

DOI: https://doi.org/10.1101/2024.08.24.608776
EMBO J. 2024 Sep 04.

Disordered regions in the IRE1α ER lumenal domain mediate its stress-induced clustering.

Paulina Kettel, Laura Marosits, Elena Spinetti, Michael Rechberger, Caterina Giannini, Philipp Radler, Isabell Niedermoser, Irmgard Fischer, Gijs A Versteeg, Martin Loose, Roberto Covino, G Elif Karagöz.

  Conserved signaling cascades monitor protein-folding homeostasis to ensure proper cellular function. One of the evolutionary conserved key players is IRE1, which maintains endoplasmic reticulum (ER) homeostasis through the unfolded protein response (UPR). Upon accumulation of misfolded proteins in the ER, IRE1 forms clusters on the ER membrane to initiate UPR signaling. What regulates IRE1 cluster formation is not fully understood. Here, we show that the ER lumenal domain (LD) of human IRE1α forms biomolecular condensates in vitro. IRE1α LD condensates were stabilized both by binding to unfolded polypeptides as well as by tethering to model membranes, suggesting their role in assembling IRE1α into signaling-competent stable clusters. Molecular dynamics simulations indicated that weak multivalent interactions drive IRE1α LD clustering. Mutagenesis experiments identified disordered regions in IRE1α LD to control its clustering in vitro and in cells. Importantly, dysregulated clustering of IRE1α mutants led to defects in IRE1α signaling. Our results revealed that disordered regions in IRE1α LD control its clustering and suggest their role as a common strategy in regulating protein assembly on membranes.

Keywords:  Biomolecular Condensates; IRE1; Supported Lipid Bilayers; Unfolded Protein Response

DOI:  https://doi.org/10.1038/s44318-024-00207-0
Proc Natl Acad Sci U S A. 2024 Sep 03. 121(36): e2408787121

Alternating binding and p97-mediated dissociation of SDS22 and I3 recycles active PP1 between holophosphatases.

Anja F Kueck, Johannes van den Boom, Sandra Koska, David Ron, Hemmo Meyer.

  Protein phosphatase-1 catalytic subunit (PP1) joins diverse targeting subunits to form holophosphatases that regulate many cellular processes. Newly synthesized PP1 is known to be transiently sequestered in an inhibitory complex with Suppressor-of-Dis2-number-2 (SDS22) and Inhibitor-3 (I3), which is disassembled by the ATPases Associated with diverse cellular Activities plus (AAA+) protein p97. Here, we show that the SDS22-PP1-I3 complex also acts as a thermodynamic sink for mature PP1 and that cycles of SDS22-PP1-I3 formation and p97-driven disassembly regulate PP1 function and subunit exchange beyond PP1 biogenesis. Förster Resonance energy transfer (FRET) analysis of labeled proteins in vitro revealed that in the p97-mediated disassembly step, both SDS22 and I3 dissociate concomitantly, releasing PP1. In presence of a targeting subunit, for instance Growth Arrest and DNA Damage-inducible protein 34 (GADD34), liberated PP1 formed an active holophosphatase that dephosphorylated its substrate, eukaryotic translation initiation factor 2 alpha (eIF2α). Inhibition of p97 results in displacement of the GADD34 targeting subunit by rebinding of PP1 to SDS22 and I3 indicating that the SDS22-PP1-I3 complex is thermodynamically favored. Likewise, p97 inhibition in cells causes rapid sequestration of PP1 by free SDS22 and I3 at the expense of other subunits. This suggests that PP1 exists in a steady state maintained by spontaneous SDS22-PP1-I3 formation and adenosine triphosphate (ATP) hydrolysis, p97-driven disassembly that recycles active PP1 between different holophosphatase complexes to warrant a dynamic holophosphatase landscape.

Keywords:  AAA+ ATPase; Förster resonance energy transfer; VCP; protein phosphatase-1; protein unfolding

DOI:  https://doi.org/10.1073/pnas.2408787121
Elife. 2024 Sep 02. pii: RP96699. [Epub ahead of print]13

Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans.

Dipti Ranjan Lenka, Shakti Virendra Dahe, Odetta Antico, Pritiranjan Sahoo, Alan R Prescott, Miratul M K Muqit, Atul Kumar.

  Loss-of-function Parkin mutations lead to early-onset of Parkinson's disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by dual phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by the PINK1 kinase. Herein, we demonstrate a competitive binding of the phospho-Ubl and RING2 domains towards the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can complex with native Parkin, leading to the activation of autoinhibited native Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, the crystal structure reveals a donor ubiquitin binding pocket in the linker connecting REP and RING2, which plays a crucial role in Parkin activity.

Keywords:  E. coli; Parkin E3 ligase; Parkinson's disease; Ubiquitin; molecular biophysics; structural biology; structure

DOI:  https://doi.org/10.7554/eLife.96699
Nat Commun. 2024 Aug 29. 15(1): 7505

Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway.

Ian Cooney, Heidi L Schubert, Karina Cedeno, Olivia N Fisher, Richard Carson, John C Price, Christopher P Hill, Peter S Shen.

The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase motor domains, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of native structures of Cdc48 affinity purified from budding yeast lysate in complex with the adaptor Shp1 in the act of unfolding substrate. Our analysis reveals a continuum of structural snapshots that spans the entire translocation cycle. These data uncover elements of Shp1-Cdc48 interactions and support a 'hand-over-hand' mechanism in which the sequential movement of individual subunits is closely coordinated. D1 hydrolyzes ATP and disengages from substrate prior to D2, while D2 rebinds ATP and re-engages with substrate prior to D1, thereby explaining the dominant role played by the D2 motor in substrate translocation/unfolding.

DOI: https://doi.org/10.1038/s41467-024-51835-3
Cell Rep. 2024 Aug 27. pii: S2211-1247(24)01040-4. [Epub ahead of print]43(9): 114689

The ULK1 effector BAG2 regulates autophagy initiation by modulating AMBRA1 localization.

Devanarayanan Siva Sankar, Stephanie Kaeser-Pebernard, Christine Vionnet, Sebastian Favre, Lais de Oliveira Marchioro, Benjamin Pillet, Jianwen Zhou, Michael Stumpe, Werner Josef Kovacs, Dieter Kressler, Manuela Antonioli, Gian Maria Fimia, Jӧrn Dengjel.

  Autophagy initiation is regulated by the ULK1 kinase complex. To gain insights into functions of the holo-complex, we generated a deep interactome by combining affinity purification- and proximity labeling-mass spectrometry of all four complex members: ULK1, ATG13, ATG101, and RB1CC1/FIP200. Under starvation conditions, the ULK1 complex interacts with several protein and lipid kinases and phosphatases, implying the formation of a signalosome. Interestingly, several selective autophagy receptors also interact with ULK1, indicating the activation of selective autophagy pathways by nutrient starvation. One effector of the ULK1 complex is the HSC/HSP70 co-chaperone BAG2, which regulates the subcellular localization of the VPS34 lipid kinase complex member AMBRA1. Depending on the nutritional status, BAG2 has opposing roles. In growth conditions, the unphosphorylated form of BAG2 sequesters AMBRA1, attenuating autophagy induction. In starvation conditions, ULK1 phosphorylates BAG2 on Ser31, which supports the recruitment of AMBRA1 to the ER membrane, positively affecting autophagy.

Keywords:  AP-MS; BioID; CP: Cell biology; CP: Molecular biology; ER; autophagy; interactome; kinase; mass spectromtery; proteomics; signaling; signalosome

DOI:  https://doi.org/10.1016/j.celrep.2024.114689
Nat Commun. 2024 Sep 03. 15(1): 7681

Multi-protein assemblies orchestrate co-translational enzymatic processing on the human ribosome.

Marius Klein, Klemens Wild, Irmgard Sinning.

Nascent chains undergo co-translational enzymatic processing as soon as their N-terminus becomes accessible at the ribosomal polypeptide tunnel exit (PTE). In eukaryotes, N-terminal methionine excision (NME) by Methionine Aminopeptidases (MAP1 and MAP2), and N-terminal acetylation (NTA) by N-Acetyl-Transferase A (NatA), is the most common combination of subsequent modifications carried out on the 80S ribosome. How these enzymatic processes are coordinated in the context of a rapidly translating ribosome has remained elusive. Here, we report two cryo-EM structures of multi-enzyme complexes assembled on vacant human 80S ribosomes, indicating two routes for NME-NTA. Both assemblies form on the 80S independent of nascent chain substrates. Irrespective of the route, NatA occupies a non-intrusive 'distal' binding site on the ribosome which does not interfere with MAP1 or MAP2 binding nor with most other ribosome-associated factors (RAFs). NatA can partake in a coordinated, dynamic assembly with MAP1 through the hydra-like chaperoning function of the abundant Nascent Polypeptide-Associated Complex (NAC). In contrast to MAP1, MAP2 completely covers the PTE and is thus incompatible with NAC and MAP1 recruitment. Together, our data provide the structural framework for the coordinated orchestration of NME and NTA in protein biogenesis.

DOI: https://doi.org/10.1038/s41467-024-51964-9
Nat Commun. 2024 Sep 02. 15(1): 7418

DMDA-PatA mediates RNA sequence-selective translation repression by anchoring eIF4A and DDX3 to GNG motifs.

Hironori Saito, Yuma Handa, Mingming Chen, Tilman Schneider-Poetsch, Yuichi Shichino, Mari Takahashi, Daniel Romo, Minoru Yoshida, Alois Fürstner, Takuhiro Ito, Kaori Fukuzawa, Shintaro Iwasaki.

Small-molecule compounds that elicit mRNA-selective translation repression have attracted interest due to their potential for expansion of druggable space. However, only a limited number of examples have been reported to date. Here, we show that desmethyl desamino pateamine A (DMDA-PatA) represses translation in an mRNA-selective manner by clamping eIF4A, a DEAD-box RNA-binding protein, onto GNG motifs. By systematically comparing multiple eIF4A inhibitors by ribosome profiling, we found that DMDA-PatA has unique mRNA selectivity for translation repression. Unbiased Bind-n-Seq reveals that DMDA-PatA-targeted eIF4A exhibits a preference for GNG motifs in an ATP-independent manner. This unusual RNA binding sterically hinders scanning by 40S ribosomes. A combination of classical molecular dynamics simulations and quantum chemical calculations, and the subsequent development of an inactive DMDA-PatA derivative reveals that the positive charge of the tertiary amine on the trienyl arm induces G selectivity. Moreover, we identified that DDX3, another DEAD-box protein, is an alternative DMDA-PatA target with the same effects on eIF4A. Our results provide an example of the sequence-selective anchoring of RNA-binding proteins and the mRNA-selective inhibition of protein synthesis by small-molecule compounds.

DOI: https://doi.org/10.1038/s41467-024-51635-9
RSC Chem Biol. 2024 Aug 28. 5(9): 866-876

Metabolically activated proteostasis regulators that protect against erastin-induced ferroptosis.

Gabriel M Kline, Nicole Madrazo, Christian M Cole, Meera Pannikkat, Michael J Bollong, Jessica D Rosarda, Jeffery W Kelly, R Luke Wiseman.

We previously showed that the proteostasis regulator compound AA147 (N-(2-hydroxy-5-methylphenyl)benzenepropanamide) potently protects against neurotoxic insults, such as glutamate-induced oxytosis. Though AA147 is a selective activator of the ATF6 arm of the unfolded protein response in non-neuronal cells, AA147-dependent protection against glutamate toxicity in cells of neuronal origin is primarily mediated through activation of the NRF2 oxidative stress response. AA147 activates NRF2 through a mechanism involving metabolic activation of AA147 by endoplasmic reticulum (ER) oxidases, affording an AA147-based quinone methide that covalently targets the NRF2 repressor protein KEAP1. Previous results show that the 2-amino-p-cresol A-ring of AA147 is required for NRF2 activation, while the phenyl B-ring of AA147 is amenable to modification. Here we explore whether the protease-sensitive amide linker between the A- and B-rings of this molecule can be modified to retain NRF2 activation. We show that replacement of the amide linker of AA147 with a carbamate linker retains NRF2 activation in neuronal cells and improves protection against neurotoxic insults, including glutamate-induced oxytosis and erastin-induced ferroptosis. Moreover, we demonstrate that inclusion of this carbamate linker facilitates identification of next-generation AA147 analogs with improved cellular tolerance and activity in disease-relevant assays.

DOI: https://doi.org/10.1039/d4cb00027g
Cell Commun Signal. 2024 Aug 30. 22(1): 421

Misfolding and aggregation in neurodegenerative diseases: protein quality control machinery as potential therapeutic clearance pathways.

Oliwia Koszła, Przemysław Sołek.

  The primary challenge in today's world of neuroscience is the search for new therapeutic possibilities for neurodegenerative disease. Central to these disorders lies among other factors, the aberrant folding, aggregation, and accumulation of proteins, resulting in the formation of toxic entities that contribute to neuronal degeneration. This review concentrates on the key proteins such as β-amyloid (Aβ), tau, and α-synuclein, elucidating the intricate molecular events underlying their misfolding and aggregation. We critically evaluate the molecular mechanisms governing the elimination of misfolded proteins, shedding light on potential therapeutic strategies. We specifically examine pathways such as the endoplasmic reticulum (ER) and unfolded protein response (UPR), chaperones, chaperone-mediated autophagy (CMA), and the intersecting signaling of Keap1-Nrf2-ARE, along with autophagy connected through p62. Above all, we emphasize the significance of these pathways as protein quality control mechanisms, encompassing interventions targeting protein aggregation, regulation of post-translational modifications, and enhancement of molecular chaperones and clearance. Additionally, we focus on current therapeutic possibilities and new, multi-target approaches. In conclusion, this review systematically consolidates insights into emerging therapeutic strategies predicated on protein aggregates clearance.

Keywords:  Aggregates; Chaperones; Misfolded proteins; Neurodegenerative disease; Tau; α-synuclein; β-amyloid

DOI:  https://doi.org/10.1186/s12964-024-01791-8
Nat Commun. 2024 Sep 05. 15(1): 7743

The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation.

Agata N Makar, Alina Boraman, Peter Mosen, Joanne E Simpson, Jair Marques, Tim Michelberger, Stuart Aitken, Ann P Wheeler, Dominic Winter, Alex von Kriegsheim, Noor Gammoh.

Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosolic components. The final degradation step is essential for clearing autophagic cargo and recycling macromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conserved transmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation.

DOI: https://doi.org/10.1038/s41467-024-52049-3
Proc Natl Acad Sci U S A. 2024 Sep 10. 121(37): e2402817121

Decoding the molecular mechanism of selective autophagy of glycogen mediated by autophagy receptor STBD1.

Yuchao Zhang, Yishan Sun, Jungang Shi, Peng Xu, Yingli Wang, Jianping Liu, Xinyu Gong, Yaru Wang, Yubin Tang, Haobo Liu, Xindi Zhou, Zhiqiao Lin, Otto Baba, Tsuyoshi Morita, Biao Yu, Lifeng Pan.

  Autophagy of glycogen (glycophagy) is crucial for the maintenance of cellular glucose homeostasis and physiology in mammals. STBD1 can serve as an autophagy receptor to mediate glycophagy by specifically recognizing glycogen and relevant key autophagic factors, but with poorly understood mechanisms. Here, we systematically characterize the interactions of STBD1 with glycogen and related saccharides, and determine the crystal structure of the STBD1 CBM20 domain with maltotetraose, uncovering a unique binding mode involving two different oligosaccharide-binding sites adopted by STBD1 CBM20 for recognizing glycogen. In addition, we demonstrate that the LC3-interacting region (LIR) motif of STBD1 can selectively bind to six mammalian ATG8 family members. We elucidate the detailed molecular mechanism underlying the selective interactions of STBD1 with ATG8 family proteins by solving the STBD1 LIR/GABARAPL1 complex structure. Importantly, our cell-based assays reveal that both the STBD1 LIR/GABARAPL1 interaction and the intact two oligosaccharide binding sites of STBD1 CBM20 are essential for the effective association of STBD1, GABARAPL1, and glycogen in cells. Finally, through mass spectrometry, biochemical, and structural modeling analyses, we unveil that STBD1 can directly bind to the Claw domain of RB1CC1 through its LIR, thereby recruiting the key autophagy initiation factor RB1CC1. In all, our findings provide mechanistic insights into the recognitions of glycogen, ATG8 family proteins, and RB1CC1 by STBD1 and shed light on the potential working mechanism of STBD1-mediated glycophagy.

Keywords:  GABARAPL1; RB1CC1; STBD1; glycogen; glycophagy

DOI:  https://doi.org/10.1073/pnas.2402817121
bioRxiv. 2024 Aug 22. pii: 2024.08.21.608176. [Epub ahead of print]

Histone deacetylase inhibition expands cellular proteostasis repertoires to enhance neuronal stress resilience.

Caitlin A Taylor, Maya Maor-Nof, Eyal Metzl-Raz, Aaron Hidalgo, Callista Yee, Aaron D Gitler, Kang Shen.

Neurons are long-lived, terminally differentiated cells with limited regenerative capacity. Cellular stressors such as endoplasmic reticulum (ER) protein folding stress and membrane trafficking stress accumulate as neurons age and accompany age-dependent neurodegeneration. Current strategies to improve neuronal resilience are focused on using factors to reprogram neurons or targeting specific proteostasis pathways. We discovered a different approach. In an unbiased screen for modifiers of neuronal membrane trafficking defects, we unexpectedly identified a role for histone deacetylases (HDACs) in limiting cellular flexibility in choosing cellular pathways to respond to diverse types of stress. We show that genetic or pharmacological inactivation of HDACs results in improved neuronal health in response to ER protein folding stress and endosomal membrane trafficking stress in C. elegans and mammalian neurons. Surprisingly, HDAC inhibition enabled neurons to activate latent proteostasis pathways tailored to the nature of the individual stress, instead of generalized transcriptional upregulation. These findings shape our understanding of neuronal stress responses and suggest new therapeutic strategies to enhance neuronal resilience.

DOI: https://doi.org/10.1101/2024.08.21.608176
Commun Biol. 2024 Sep 04. 7(1): 1083

Differential effects of 40S ribosome recycling factors on reinitiation at regulatory uORFs in GCN4 mRNA are not dictated by their roles in bulk 40S recycling.

Kristína Jendruchová, Swati Gaikwad, Kristýna Poncová, Stanislava Gunišová, Leoš Shivaya Valášek, Alan G Hinnebusch.

Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation (REI) at short upstream open reading frames (uORFs) harboring penultimate codons that confer heightened dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited REI at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on REI at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) REI. We found that the Tma proteins generally impede REI at native uORF4 and its variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on REI at native uORF1 and equipping it with Tma-hyperdependent penultimate codons generally did not confer Tma-dependent REI; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the REI potential of the uORF and penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.

DOI: https://doi.org/10.1038/s42003-024-06761-x
Nucleic Acids Res. 2024 Sep 03. pii: gkae768. [Epub ahead of print]

PROTAC-DB 3.0: an updated database of PROTACs with extended pharmacokinetic parameters.

Jingxuan Ge, Shimeng Li, Gaoqi Weng, Huating Wang, Meijing Fang, Huiyong Sun, Yafeng Deng, Chang-Yu Hsieh, Dan Li, Tingjun Hou.

Proteolysis-targeting chimera (PROTAC) is an emerging therapeutic technology that leverages the ubiquitin-proteasome system to target protein degradation. Due to its event-driven mechanistic characteristics, PROTAC has the potential to regulate traditionally non-druggable targets. Recently, AI-aided drug design has accelerated the development of PROTAC drugs. However, the rational design of PROTACs remains a considerable challenge. Here, we present an updated online database, PROTAC-DB 3.0. In this third version, we have expanded the database to include 6111 PROTACs (87% increase compared to the 2.0 version). Additionally, the database now contains 569 warheads (small molecules targeting the protein), 2753 linkers, and 107 E3 ligands (small molecules recruiting E3 ligases). The number of target-PROTAC-E3 ternary complex structures has also increased to 959. Recognizing the importance of druggability in PROTAC design, we have incorporated pharmacokinetic data to PROTAC-DB 3.0. To enhance user experience, we have added features for sorting based on molecular similarity and literature publication date. PROTAC-DB 3.0 is accessible at http://cadd.zju.edu.cn/protacdb/.

DOI: https://doi.org/10.1093/nar/gkae768
Science. 2024 Aug 30. 385(6712): 1009-1016

Aggregate-selective removal of pathological tau by clustering-activated degraders.

Jonathan Benn, Shi Cheng, Sophie Keeling, Annabel E Smith, Marina J Vaysburd, Dorothea Böken, Lauren V C Miller, Taxiarchis Katsinelos, Catarina Franco, Elian Dupré, Clément Danis, Isabelle Landrieu, Luc Buée, David Klenerman, Leo C James, William A McEwan.

Selective degradation of pathological protein aggregates while sparing monomeric forms is of major therapeutic interest. The E3 ligase tripartite motif-containing protein 21 (TRIM21) degrades antibody-bound proteins in an assembly state-specific manner due to the requirement of TRIM21 RING domain clustering for activation, yet effective targeting of intracellular assemblies remains challenging. Here, we fused the RING domain of TRIM21 to a target-specific nanobody to create intracellularly expressed constructs capable of selectively degrading assembled proteins. We evaluated this approach against green fluorescent protein-tagged histone 2B (H2B-GFP) and tau, a protein that undergoes pathological aggregation in Alzheimer's and other neurodegenerative diseases. RING-nanobody degraders prevented or reversed tau aggregation in culture and in vivo, with minimal impact on monomeric tau. This approach may have therapeutic potential for the many disorders driven by intracellular protein aggregation.

DOI: https://doi.org/10.1126/science.adp5186
Nat Chem Biol. 2024 Aug 30.

µMap proximity labeling in living cells reveals stress granule disassembly mechanisms.

Chenmengxiao Roderick Pan, Steve D Knutson, Sean W Huth, David W C MacMillan.

Phase-separated condensates are membrane-less intracellular structures comprising dynamic protein interactions that organize essential biological processes. Understanding the composition and dynamics of these organelles advances our knowledge of cellular behaviors and disease pathologies related to granule dysregulation. In this study, we apply microenvironment mapping with a HaloTag-based platform (HaloMap) to characterize intracellular stress granule dynamics in living cells. After validating the robustness and sensitivity of this approach, we then profile the stress granule proteome throughout the formation and disassembly and under pharmacological perturbation. These experiments reveal several ubiquitin-related modulators, including the HECT (homologous to E6AP C terminus) E3 ligases ITCH and NEDD4L, as well as the ubiquitin receptor toll-interacting protein TOLLIP, as key mediators of granule disassembly. In addition, we identify an autophagy-related pathway that promotes granule clearance. Collectively, this work establishes a general photoproximity labeling approach for unraveling intracellular protein interactomes and uncovers previously unexplored regulatory mechanisms of stress granule dynamics.

DOI: https://doi.org/10.1038/s41589-024-01721-2
Nat Cell Biol. 2024 Aug 29.

Nuclear proteasomes buffer cytoplasmic proteins during autophagy compromise.

So Jung Park, Sung Min Son, Antonio Daniel Barbosa, Lidia Wrobel, Eleanna Stamatakou, Ferdinando Squitieri, Gabriel Balmus, David C Rubinsztein.

Autophagy is a conserved pathway where cytoplasmic contents are engulfed by autophagosomes, which then fuse with lysosomes enabling their degradation. Mutations in core autophagy genes cause neurological conditions, and autophagy defects are seen in neurodegenerative diseases such as Parkinson's disease and Huntington's disease. Thus, we have sought to understand the cellular pathway perturbations that autophagy-perturbed cells are vulnerable to by seeking negative genetic interactions such as synthetic lethality in autophagy-null human cells using available data from yeast screens. These revealed that loss of proteasome and nuclear pore complex components cause synergistic viability changes akin to synthetic fitness loss in autophagy-null cells. This can be attributed to the cytoplasm-to-nuclear transport of proteins during autophagy deficiency and subsequent degradation of these erstwhile cytoplasmic proteins by nuclear proteasomes. As both autophagy and cytoplasm-to-nuclear transport are defective in Huntington's disease, such cells are more vulnerable to perturbations of proteostasis due to these synthetic interactions.

DOI: https://doi.org/10.1038/s41556-024-01488-7
J Biol Chem. 2024 Aug 29. pii: S0021-9258(24)02232-4. [Epub ahead of print] 107731

Delineation of the substrate recognition domain of MARCHF6 E3 ubiquitin ligase in the Ac/N-degron pathway and its regulatory role in ferroptosis.

Jihye Yang, Sang-Yoon Kim, Cheol-Sang Hwang.

  Nα-terminal acetylation in eukaryotic proteins creates specific degradation signals (Ac/N-degrons) targeted for ubiquitin-mediated proteolysis via the Ac/N-degron pathway. Despite the identification of key components of the Ac/N-degron pathway over the past 15 years, the precise recognition domain (Ac/N domain) remains unclear. Here, we defined the Ac/N domain of the endoplasmic reticulum MARCHF6 E3 ubiquitin ligase through a systematic analysis of its cytosol-facing regions using alanine-stretch mutagenesis, chemical crosslinking-based co-immunoprecipitation-immunoblotting, and split-ubiquitin assays in human and yeast cells. The Ac/N domain of MARCHF6 exhibits preferential binding specificity to Nα-terminally acetylated proteins and peptides over their unacetylated counterparts, mediating the degradation of Ac/N-degron-bearing proteins, such as the G-protein regulator RGS2 and the lipid droplet protein PLIN2. Furthermore, abolishing the recognition of Ac/N-degrons by MARCHF6 stabilized RGS2 and PLIN2, thereby increasing the resistance to ferroptosis, an iron-dependent lipid peroxidation-mediated cell death. These findings provide mechanistic and functional insights into how MARCHF6 serves as a rheostatic modulator of ferroptosis by recognizing Ac/N-degron substrates via its Ac/N domain and non-Ac/N-degron substrates via distinct recognition sites.

Keywords:  Acetylation/N domain; MARCHF6; N-terminal acetylation; degradation signal; ferroptosis; proteolysis; ubiquitin

DOI:  https://doi.org/10.1016/j.jbc.2024.107731
Proc Natl Acad Sci U S A. 2024 Sep 10. 121(37): e2403038121

ER-associated degradation ligase HRD1 links ER stress to DNA damage repair by modulating the activity of DNA-PKcs.

Zhiyuan Xiang, Guixue Hou, Shanliang Zheng, Minqiao Lu, Tianyu Li, Qingyu Lin, Hao Liu, Xingwen Wang, Tianqi Guan, Yuhan Wei, Wenxin Zhang, Yi Zhang, Chaoran Liu, Li Li, Qun-Ying Lei, Ying Hu.

  Proteostasis and genomic integrity are respectively regulated by the endoplasmic reticulum-associated protein degradation (ERAD) and DNA damage repair signaling pathways, with both pathways essential for carcinogenesis and drug resistance. How these signaling pathways coordinate with each other remains unexplored. We found that ER stress specifically induces the DNA-PKcs-regulated nonhomologous end joining (NHEJ) pathway to amend DNA damage and impede cell death. Intriguingly, sustained ER stress rapidly decreased the activity of DNA-PKcs and DNA damage accumulated, facilitating a switch from adaptation to cell death. This DNA-PKcs inactivation was caused by increased KU70/KU80 protein degradation. Unexpectedly, the ERAD ligase HRD1 was found to efficiently destabilize the classic nuclear protein HDAC1 in the cytoplasm, by catalyzing HDAC1's polyubiquitination at lysine 74, at a late stage of ER stress. By abolishing HDAC1-mediated KU70/KU80 deacetylation, HRD1 transmits ER signals to the nucleus. The resulting enhanced KU70/KU80 acetylation provides binding sites for the nuclear E3 ligase TRIM25, resulting in the promotion of polyubiquitination and the degradation of KU70/KU80 proteins. Both in vitro and in vivo cancer models showed that genetic or pharmacological inhibition of HADC1 or DNA-PKcs sensitizes colon cancer cells to ER stress inducers, including the Food and Drug Administration-approved drug celecoxib. The antitumor effects of the combined approach were also observed in patient-derived xenograft models. These findings identify a mechanistic link between ER stress (ERAD) in the cytoplasm and DNA damage (NHEJ) pathways in the nucleus, indicating that combined anticancer strategies may be developed that induce severe ER stress while simultaneously inhibiting KU70/KU80/DNA-PKcs-mediated NHEJ signaling.

Keywords:  DNA damage; HDAC1; KU70/KU80; TRIM25; endoplasmic reticulum stress

DOI:  https://doi.org/10.1073/pnas.2403038121
Cell Rep. 2024 Aug 29. pii: S2211-1247(24)01038-6. [Epub ahead of print]43(9): 114687

E3 ubiquitin ligase ANKIB1 attenuates antiviral immune responses by promoting K48-linked polyubiquitination of MAVS.

Wei Liu, Cui Yuan, Buwen Fu, Jiufeng Xie, Wenqing Li, Guozhi Zhang, Zhenling Ma, Pengtao Jiao.

  Upon sensing cytosolic viral RNA, retinoic acid-inducible gene-I-like receptors (RLRs) interact with mitochondrial antiviral signaling proteins (MAVSs) to activate IRF3 and nuclear factor κB (NF-κB) signaling, initiating innate immune responses. Thus, RLR activation plays a vital role in the removal of invasive RNA viruses while maintaining immune homeostasis. However, inadequate or excessive activation of immunity can cause harm and can even lead to lethal consequences. In this study, we identify an E3 ligase, ankyrin repeat and IBR domain containing 1 (ANKIB1), which suppresses RLR signaling via MAVS. ANKIB1 binds to MAVS to enhance K48-linked polyubiquitination with K311R, causing proteasomal degradation of MAVS. Deficiency of ANKIB1 significantly increases the RLR-mediated production of type I interferon (IFN) along with pro-inflammatory factors. Consequently, ANKIB1 deficiency remarkably increases antiviral immunity and decreases viral replication in vivo. Therefore, we reveal that ANKIB1 restricts RLR-induced innate immune activation, indicating its potential role as a therapeutic target for viral infections.

Keywords:  ANKIB1; CP: Immunology; MAVS; RLR; innate immunity; type I interferons

DOI:  https://doi.org/10.1016/j.celrep.2024.114687
JCI Insight. 2024 Aug 29. pii: e179525. [Epub ahead of print]

Mutant induced neurons and humanized mice enable identification of Niemann-Pick C1 proteostatic therapies.

Ruth D Azaria, Adele B Correia, Kylie J Schache, Manuela Zapata, Koralege C Pathmasiri, Varshasnata Mohanty, Dharma T Nannapaneni, Brandon L Ashfeld, Paul Helquist, Olaf Wiest, Kenji Ohgane, Qingqing Li, Ross A Fredenburg, Brian Sj Blagg, Stephanie M Cologna, Mark L Schultz, Andrew P Lieberman.

  Therapeutics that rescue folding, trafficking, and function of disease-causing missense mutants are sought for a host of human diseases, but efforts to leverage model systems to test emerging strategies have met with limited success. Such is the case for Niemann-Pick type C1 disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, progressive neurodegeneration, and early death. NPC1, a multipass transmembrane glycoprotein, is synthesized in the endoplasmic reticulum and traffics to late endosomes/lysosomes, but this process is often disrupted in disease. We sought to identify small molecules that promote folding and enable lysosomal localization and functional recovery of mutant NPC1. We leveraged a panel of isogenic human induced neurons expressing distinct NPC1 missense mutations. We used this panel to rescreen compounds that were reported previously to correct NPC1 folding and trafficking. We established mo56-hydroxycholesterol (mo56Hc) as a potent pharmacological chaperone for several NPC1 mutants. Furthermore, we generated mice expressing human I1061T NPC1, a common mutation in patients. We demonstrated that this model exhibited disease phenotypes and recapitulated the protein trafficking defects, lipid storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a paradigm for testing and validation of proteostatic therapeutics as an important step towards the development of disease-modifying therapies.

Keywords:  Lysosomes; Neurological disorders; Neuroscience; Protein misfolding

DOI:  https://doi.org/10.1172/jci.insight.179525
Nat Commun. 2024 Aug 31. 15(1): 7596

Leveraging multiple data types for improved compound-kinase bioactivity prediction.

Ryan Theisen, Tianduanyi Wang, Balaguru Ravikumar, Rayees Rahman, Anna Cichońska.

Machine learning provides efficient ways to map compound-kinase interactions. However, diverse bioactivity data types, including single-dose and multi-dose-response assay results, present challenges. Traditional models utilize only multi-dose data, overlooking information contained in single-dose measurements. Here, we propose a machine learning methodology for compound-kinase activity prediction that leverages both single-dose and dose-response data. We demonstrate that our two-stage approach yields accurate activity predictions and significantly improves model performance compared to training solely on dose-response labels. This superior performance is consistent across five diverse machine learning methods. Using the best performing model, we carried out extensive experimental profiling on a total of 347 selected compound-kinase pairs, achieving a high hit rate of 40% and a negative predictive value of 78%. We show that these rates can be improved further by incorporating model uncertainty estimates into the compound selection process. By integrating multiple activity data types, we demonstrate that our approach holds promise for facilitating the development of training activity datasets in a more efficient and cost-effective way.

DOI: https://doi.org/10.1038/s41467-024-52055-5
bioRxiv. 2024 Aug 07. pii: 2024.08.06.606767. [Epub ahead of print]

Episodic Transport of Protein Aggregates Achieves a Positive Size Selectivity in Aggresome Formation.

Rui Fang, Luolan Bai, Boyan Li, Kevin Dong, Joao A Paulo, Mengying Zhou, Yi-Chi Chu, Yuyu Song, Michael Y Sherman, Steven Gygi, Christine M Field, Timothy J Mitchison, Ying Lu.

Eukaryotic cells direct toxic misfolded proteins to various protein quality control pathways based on their chemical features and aggregation status. Aggregated proteins are targeted to selective autophagy or specifically sequestered into the "aggresome," a perinuclear inclusion at the microtubule-organizing center (MTOC). However, the mechanism for selectively sequestering protein aggregates into the aggresome remains unclear. To investigate aggresome formation, we reconstituted MTOC-directed aggregate transport in Xenopus laevis egg extract using AgDD, a chemically inducible aggregation system. High-resolution single-particle tracking revealed that dynein-mediated transport of aggregates was highly episodic, with average velocity positively correlated with aggregate size. Our mechanistic model suggests that the recurrent formation of the dynein transport complex biases larger aggregates towards the active transport state, compensating for the slowdown due to viscosity. Both episodic transport and positive size selectivity are specifically associated with aggresome-dynein adaptors. Coupling conventional dynein-activating adaptors to the aggregates perturbs aggresome formation and reverses size selectivity.

DOI: https://doi.org/10.1101/2024.08.06.606767
Autophagy. 2024 Sep 03. 1-3

Nucleoid-phagy: a novel safeguard against mitochondrial DNA-Induced inflammation.

Hao Liu, Jianming Xie, Cien Zhen, Lin Zeng, Hualin Fan, Haixia Zhuang, Du Feng.

  Mitochondria, the powerhouses of the cell, play pivotal roles in cellular processes ranging from energy production to innate immunity. Their unique double-membrane structure typically sequesters mitochondrial DNA (mtDNA) from the rest of the cell. However, under oxidative or immune stress, mtDNA can escape into the cytoplasm, posing a threat as a potential danger signal. The accumulation of cytoplasmic mtDNA can disrupt cellular immune balance and trigger cell death. Our research unveils a novel quality control mechanism, which we term "nucleoid-phagy", that safeguards cellular homeostasis by clearing mislocalized mtDNA. We demonstrate that TFAM, a key protein involved in mtDNA folding and wrapping, accompanies mtDNA into the cytoplasm under stress conditions. Remarkably, TFAM acts as an autophagy receptor, interacting with LC3B to facilitate the autophagic clearance of cytoplasmic mtDNA, thereby preventing the activation of the pro-inflammatory CGAS-STING1 pathway. This study provides unprecedented insights into cytoplasmic mtDNA quality control and offers new perspectives on mitigating inflammatory responses in mitochondrial-related diseases.

Keywords:  Autophagy; CGAS-STING1; LIR; TFAM; mitochondria DNA

DOI:  https://doi.org/10.1080/15548627.2024.2395145
Science. 2024 Aug 30. 385(6712): eadj7446

A p62-dependent rheostat dictates micronuclei catastrophe and chromosome rearrangements.

Sara Martin, Simone Scorzoni, Sara Cordone, Alice Mazzagatti, Galina V Beznoussenko, Amanda L Gunn, Melody Di Bona, Yonatan Eliezer, Gil Leor, Tal Ben-Yishay, Alessia Loffreda, Valeria Cancila, Maria Chiara Rainone, Marica Rosaria Ippolito, Valentino Martis, Fabio Bedin, Massimiliano Garrè, Laura Pontano Vaites, Paolo Vasapolli, Simona Polo, Dario Parazzoli, Claudio Tripodo, Alexander A Mironov, Alessandro Cuomo, Uri Ben-David, Samuel F Bakhoum, Emily M Hatch, Peter Ly, Stefano Santaguida.

Chromosomal instability (CIN) generates micronuclei-aberrant extranuclear structures that catalyze the acquisition of complex chromosomal rearrangements present in cancer. Micronuclei are characterized by persistent DNA damage and catastrophic nuclear envelope collapse, which exposes DNA to the cytoplasm. We found that the autophagic receptor p62/SQSTM1 modulates micronuclear stability, influencing chromosome fragmentation and rearrangements. Mechanistically, proximity of micronuclei to mitochondria led to oxidation-driven homo-oligomerization of p62, limiting endosomal sorting complex required for transport (ESCRT)-dependent micronuclear envelope repair by triggering autophagic degradation. We also found that p62 levels correlate with increased chromothripsis across human cancer cell lines and with increased CIN in colorectal tumors. Thus, p62 acts as a regulator of micronuclei and may serve as a prognostic marker for tumors with high CIN.

DOI: https://doi.org/10.1126/science.adj7446
Mol Cell. 2024 Aug 27. pii: S1097-2765(24)00671-3. [Epub ahead of print]

Disorder-mediated interactions target proteins to specific condensates.

Nancy De La Cruz, Prashant Pradhan, Reshma T Veettil, Brooke A Conti, Mariano Oppikofer, Benjamin R Sabari.

  Selective compartmentalization of cellular contents is fundamental to the regulation of biochemistry. Although membrane-bound organelles control composition by using a semi-permeable barrier, biomolecular condensates rely on interactions among constituents to determine composition. Condensates are formed by dynamic multivalent interactions, often involving intrinsically disordered regions (IDRs) of proteins, yet whether distinct compositions can arise from these dynamic interactions is not known. Here, by comparative analysis of proteins differentially partitioned by two different condensates, we find that distinct compositions arise through specific IDR-mediated interactions. The IDRs of differentially partitioned proteins are necessary and sufficient for selective partitioning. Distinct sequence features are required for IDRs to partition, and swapping these sequence features changes the specificity of partitioning. Swapping whole IDRs retargets proteins and their biochemical activity to different condensates. Our results demonstrate that IDR-mediated interactions can target proteins to specific condensates, enabling the spatial regulation of biochemistry within the cell.

Keywords:  IDR; biomolecular condensates; condensate composition; condensate function; intrinsically disordered regions; nuclear organization; specificity

DOI:  https://doi.org/10.1016/j.molcel.2024.08.017
bioRxiv. 2024 Aug 24. pii: 2024.08.20.608660. [Epub ahead of print]

Lipid- and protein-directed photosensitizer proximity labeling captures the cholesterol interactome.

Andrew P Becker, Elijah Biletch, John Paul Kennelly, Ashley R Julio, Miranda Villaneuva, Rohith T Nagari, Daniel W Turner, Nikolas R Burton, Tomoyuki Fukuta, Liujuan Cui, Xu Xiao, Soon-Gook Hong, Julia J Mack, Peter Tontonoz, Keriann M Backus.

The physical properties of cellular membranes, including fluidity and function, are influenced by protein and lipid interactions. In situ labeling chemistries, most notably proximity-labeling interactomics are well suited to characterize these dynamic and often fleeting interactions. Established methods require distinct chemistries for proteins and lipids, which limits the scope of such studies. Here we establish a singlet-oxygen-based photocatalytic proximity labeling platform (POCA) that reports intracellular interactomes for both proteins and lipids with tight spatiotemporal resolution using cell-penetrant photosensitizer reagents. Using both physiologically relevant lipoprotein-complexed probe delivery and genetic manipulation of cellular cholesterol handling machinery, cholesterol-directed POCA captured established and unprecedented cholesterol binding proteins, including protein complexes sensitive to intracellular cholesterol levels and proteins uniquely captured by lipoprotein uptake. Protein-directed POCA accurately mapped known intracellular membrane complexes, defined sterol-dependent changes to the non-vesicular cholesterol transport protein interactome, and captured state-dependent changes in the interactome of the cholesterol transport protein Aster-B. More broadly, we find that POCA is a versatile interactomics platform that is straightforward to implement, using the readily available HaloTag system, and fulfills unmet needs in intracellular singlet oxygen-based proximity labeling proteomics. Thus, we expect widespread utility for POCA across a range of interactome applications, spanning imaging to proteomics.

DOI: https://doi.org/10.1101/2024.08.20.608660
Elife. 2024 Sep 04. pii: RP93256. [Epub ahead of print]13

Quantitative mapping of proteasome interactomes and substrates using ProteasomeID.

Aleksandar Bartolome, Julia C Heiby, Domenico Di Fraia, Ivonne Heinze, Hannah Knaudt, Ellen Spaeth, Omid Omrani, Alberto Minetti, Maleen Hofmann, Joanna M Kirkpatrick, Therese Dau, Alessandro Ori.

  Proteasomes are essential molecular machines responsible for the degradation of proteins in eukaryotic cells. Altered proteasome activity has been linked to neurodegeneration, auto-immune disorders and cancer. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome composition and interactions in vivo in animal models. To fill this gap, we developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and generated a new mouse model enabling the quantification of proteasome interactions by mass spectrometry. We show that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on their activity. We demonstrate the utility of our method by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling enables the identification of both endogenous and small-molecule-induced proteasome substrates.

Keywords:  biochemistry; chemical biology; human; mass spectrometry; mouse; proteasome; protein degradation; proteomics; proximity labelling

DOI:  https://doi.org/10.7554/eLife.93256
J Proteome Res. 2024 Sep 05.

Proteome and Glycoproteome Analyses Reveal Regulation of Protein Glycosylation Site-Specific Occupancy and Lysosomal Hydrolase Maturation by N-Glycan-Dependent ER-Quality Control.

Jingru Chen, Piaopiao Wen, Yu-He Tang, Hanjie Li, Zibo Wang, Xiuyuan Wang, Xiaoman Zhou, Xiao-Dong Gao, Morihisa Fujita, Ganglong Yang.

  N-Glycan-dependent endoplasmic reticulum quality control (ERQC) primarily mediates protein folding, which determines the fate of the polypeptide. Monoglucose residues on N-glycans determine whether the nascent N-glycosylated proteins enter into and escape from the calnexin (CANX)/calreticulin (CALR) cycle, which is a central system of the ERQC. To reveal the impact of ERQC on glycosylation and protein fate, we performed comprehensive quantitative proteomic and glycoproteomic analyses using cells defective in N-glycan-dependent ERQC. Deficiency of MOGS encoding the ER α-glucosidase I, CANX, or/and CALR broadly affected protein expression and glycosylation. Among the altered glycoproteins, the occupancy of oligomannosidic N-glycans was significantly affected. Besides the expected ER stress, proteins and glycoproteins involved in pathways for lysosome and viral infection are differentially changed in those deficient cells. We demonstrated that lysosomal hydrolases were not correctly modified with mannose-6-phosphates on the N-glycans and were directly secreted to the culture medium in N-glycan-dependent ERQC mutant cells. Overall, the CANX/CALR cycle promotes the correct folding of glycosylated peptides and influences the transport of lysosomal hydrolases.

Keywords:  ER-quality control; N-linked glycosylation; lysosome disorders; site-specific glycoproteome

DOI:  https://doi.org/10.1021/acs.jproteome.4c00378
Nat Cell Biol. 2024 Sep 02.

The stress response regulator HSF1 modulates natural killer cell anti-tumour immunity.

Kathryn Hockemeyer, Theodore Sakellaropoulos, Xufeng Chen, Olha Ivashkiv, Maria Sirenko, Hua Zhou, Giovanni Gambi, Elena Battistello, Kleopatra Avrampou, Zhengxi Sun, Maria Guillamot, Luis Chiriboga, George Jour, Igor Dolgalev, Kate Corrigan, Kamala Bhatt, Iman Osman, Aristotelis Tsirigos, Nikos Kourtis, Iannis Aifantis.

Diverse cellular insults converge on activation of the heat shock factor 1 (HSF1), which regulates the proteotoxic stress response to maintain protein homoeostasis. HSF1 regulates numerous gene programmes beyond the proteotoxic stress response in a cell-type- and context-specific manner to promote malignancy. However, the role(s) of HSF1 in immune populations of the tumour microenvironment remain elusive. Here, we leverage an in vivo model of HSF1 activation and single-cell transcriptomic tumour profiling to show that augmented HSF1 activity in natural killer (NK) cells impairs cytotoxicity, cytokine production and subsequent anti-tumour immunity. Mechanistically, HSF1 directly binds and regulates the expression of key mediators of NK cell effector function. This work demonstrates that HSF1 regulates the immune response under the stress conditions of the tumour microenvironment. These findings have important implications for enhancing the efficacy of adoptive NK cell therapies and for designing combinatorial strategies including modulators of NK cell-mediated tumour killing.

DOI: https://doi.org/10.1038/s41556-024-01490-z
FASEB J. 2024 Sep 15. 38(17): e70021

Promotion of endoplasmic reticulum retrotranslocation by overexpression of E3 ubiquitin-protein ligase synoviolin 1 reduces endoplasmic reticulum stress and preserves cone photoreceptors in cyclic nucleotide-gated channel deficiency.

Fan Yang, Hongwei Ma, Sanford L Boye, Shannon E Boye, Xi-Qin Ding.

  Cone photoreceptor cyclic nucleotide-gated (CNG) channels play an essential role in phototransduction and cellular Ca2+ homeostasis. Mutations in genes encoding the channel subunits CNGA3 and CNGB3 are associated with achromatopsia, progressive cone dystrophy, and early-onset macular degeneration. Cone loss in patients with achromatopsia and cone dystrophy associated with CNG channel mutations has been documented by optical coherence tomography and in mouse models of CNG channel deficiency. Cone death in CNG channel-deficient retinas involves endoplasmic reticulum (ER) stress-associated apoptosis, dysregulation of cellular/ER Ca2+ homeostasis, impaired protein folding/processing, and impaired ER-associated degradation (ERAD). The E3 ubiquitin-protein ligase synoviolin 1 (SYVN1) is the primary component of the SYVN1/SEL1L ER retrotranslocon responsible for ERAD. Previous studies have shown that manipulations that protect cones and reduce ER stress/cone death in CNG channel deficiency, such as increasing ER Ca2+ preservation or treatment with an ER chaperone, increase the expression of SYVN1 and other components of the ER retrotranslocon. The present work investigated the effects of SYVN1 overexpression. Intraocular injection of AAV5-IRBP/GNAT2-Syvn1 resulted in overexpression of SYVN1 in cones of CNG channel-deficient mice. Following treatment, cone density in Cnga3-/- mice was significantly increased, compared with untreated controls, outer segment localization of cone opsin was improved, and ER stress/apoptotic cell death was reduced. Overexpression of SYVN1 also led to increased expression levels of the retrotranslocon components, degradation in ER protein 1 (DERL1), ERAD E3 ligase adaptor subunit (SEL1L), and homocysteine inducible ER protein with ubiquitin-like domain 1 (HERPUD1). Moreover, overexpression of SYVN1 likely enhanced protein ubiquitination/proteasome degradation in CNG channel-deficient retinas. This study demonstrates the role of SYVN1/ERAD in cone preservation in CNG channel deficiency and supports the strategy of promoting ERAD for cone protection.

Keywords:  CNG channel; ER stress; ERAD; SYVN1; cone photoreceptors; retinal degeneration

DOI:  https://doi.org/10.1096/fj.202400198R
PNAS Nexus. 2024 Sep;3(9): pgae356

Identification and mechanism of wheat protein disulfide isomerase-promoted gluten network formation.

Jihui Gao, Jiayin Ma, Peixuan Yu, Dong Yang.

  Formation of the gluten network depends on glutenin crosslinking via disulfide bonds, and wheat protein disulfide isomerase (wPDI) plays an important role in this process. Here, we identify a substrate gluten protein of wPDI and the mechanism underlying wPDI-promoted glutenin crosslinking. Farinographic, rheologic, and alveographic analysis unambiguously proves that wPDI improves gluten network formation, which is directly observed by 3D reconstruction of the gluten network. Protein analysis and LC-MS/MS reveal that glutenin subunit 1Dx5 is primarily recruited by wPDI to participate in gluten network formation, and its cysteine-containing N-terminal domain (1Dx5-NTD), which harbors three cysteine residues for crosslinking, is purified. 1Dx5-NTD interacts with wPDI in both redox states, possibly folded by reduced wPDI and then catalyzed by oxidized wPDI, as further evidenced by wPDI-promoted self-crosslinking. Consistent with macroscopic observations, our results suggest that wPDI folds 1Dx5-NTD into β-strand structure that favors disulfide bond formation.

Keywords:  chaperone; crosslinking; gluten network; protein folding; wheat protein disulfide isomerase

DOI:  https://doi.org/10.1093/pnasnexus/pgae356
bioRxiv. 2024 Aug 07. pii: 2024.08.05.606646. [Epub ahead of print]

High-grade serous ovarian cancer development and anti-PD-1 resistance is driven by IRE1α activity in neutrophils.

Alexander Emmanuelli, Camilla Salvagno, Sung Min-Hwang, Deepika Awasthi, Tito A Sandoval, Chang-Suk Chae, Jin-Gyu Cheong, Chen Tan, Takao Iwawaki, Juan R Cubillos-Ruiz.

High-grade serous ovarian cancer (HGSOC) is an aggressive malignancy that remains refractory to current immunotherapies. While advanced stage disease has been extensively studied, the cellular and molecular mechanisms that promote early immune escape in HGSOC remain largely unexplored. Here we report that primary HGSO tumors program neutrophils to inhibit T cell anti-tumor function by activating the endoplasmic reticulum (ER) stress sensor IRE1α. We found that intratumoral neutrophils exhibited overactivation of ER stress response markers compared with their counterparts at non-tumor sites. Selective deletion of IRE1α in neutrophils delayed primary ovarian tumor growth and extended the survival of mice with HGSOC by enabling early T cell-mediated tumor control. Notably, loss of IRE1α in neutrophils sensitized tumor-bearing mice to PD-1 blockade, inducing HGSOC regression and long-term survival in ∼50% of treated hosts. Hence, neutrophil-intrinsic IRE1α facilitates early adaptive immune escape in HGSOC and targeting this ER stress sensor might be used to unleash endogenous and immunotherapy-elicited immunity that controls metastatic disease.

DOI: https://doi.org/10.1101/2024.08.05.606646
Life Sci Alliance. 2024 Nov;pii: e202402681. [Epub ahead of print]7(11):

Regulation of proteostasis by sleep through autophagy in Drosophila models of Alzheimer's disease.

Natalie Ortiz-Vega, Amanda G Lobato, Tijana Canic, Yi Zhu, Stanislav Lazopulo, Sheyum Syed, R Grace Zhai.

Sleep and circadian rhythm dysfunctions are common clinical features of Alzheimer's disease (AD). Increasing evidence suggests that in addition to being a symptom, sleep disturbances can also drive the progression of neurodegeneration. Protein aggregation is a pathological hallmark of AD; however, the molecular pathways behind how sleep affects protein homeostasis remain elusive. Here we demonstrate that sleep modulation influences proteostasis and the progression of neurodegeneration in Drosophila models of tauopathy. We show that sleep deprivation enhanced Tau aggregational toxicity resulting in exacerbated synaptic degeneration. In contrast, sleep induction using gaboxadol led to reduced toxic Tau accumulation in neurons as a result of modulated autophagic flux and enhanced clearance of ubiquitinated Tau, suggesting altered protein processing and clearance that resulted in improved synaptic integrity and function. These findings highlight the complex relationship between sleep and regulation of protein homeostasis and the neuroprotective potential of sleep-enhancing therapeutics to slow the progression or delay the onset of neurodegeneration.

DOI: https://doi.org/10.26508/lsa.202402681
Science. 2024 Aug 30. 385(6712): eadj8691

Micronuclear collapse from oxidative damage.

Melody Di Bona, Yanyang Chen, Albert S Agustinus, Alice Mazzagatti, Mercedes A Duran, Matthew Deyell, Daniel Bronder, James Hickling, Christy Hong, Lorenzo Scipioni, Giulia Tedeschi, Sara Martin, Jun Li, Aušrinė Ruzgaitė, Nadeem Riaz, Parin Shah, Edridge K D'Souza, D Zack Brodtman, Simone Sidoli, Bill Diplas, Manisha Jalan, Nancy Y Lee, Alban Ordureau, Benjamin Izar, Ashley M Laughney, Simon Powell, Enrico Gratton, Stefano Santaguida, John Maciejowski, Peter Ly, Thomas M Jeitner, Samuel F Bakhoum.

Chromosome-containing micronuclei are a hallmark of aggressive cancers. Micronuclei frequently undergo irreversible collapse, exposing their enclosed chromatin to the cytosol. Micronuclear rupture catalyzes chromosomal rearrangements, epigenetic abnormalities, and inflammation, yet mechanisms safeguarding micronuclear integrity are poorly understood. In this study, we found that mitochondria-derived reactive oxygen species (ROS) disrupt micronuclei by promoting a noncanonical function of charged multivesicular body protein 7 (CHMP7), a scaffolding protein for the membrane repair complex known as endosomal sorting complex required for transport III (ESCRT-III). ROS retained CHMP7 in micronuclei while disrupting its interaction with other ESCRT-III components. ROS-induced cysteine oxidation stimulated CHMP7 oligomerization and binding to the nuclear membrane protein LEMD2, disrupting micronuclear envelopes. Furthermore, this ROS-CHMP7 pathological axis engendered chromosome shattering known to result from micronuclear rupture. It also mediated micronuclear disintegrity under hypoxic conditions, linking tumor hypoxia with downstream processes driving cancer progression.

DOI: https://doi.org/10.1126/science.adj8691
J Med Chem. 2024 Sep 04.

Heat Shock Protein 90 Interactome-Mediated Proteolysis Targeting Chimera (HIM-PROTAC) Degrading Glutathione Peroxidase 4 to Trigger Ferroptosis.

Jinyun Dong, Furong Ma, Maohua Cai, Fei Cao, Haobin Li, Hui Liang, Yulong Li, Guangyu Ding, Juan Li, Xiangdong Cheng, Jiang-Jiang Qin.

Targeted protein degradation (TPD) is an emerging therapeutic paradigm aimed at eliminating the disease-causing protein with aberrant expression. Herein, we report a new approach to inducing intracellular glutathione peroxidase 4 (GPX4) protein degradation to trigger ferroptosis by bridging the target protein to heat shock protein 90 (HSP90), termed HSP90 interactome-mediated proteolysis targeting chimera (HIM-PROTAC). Different series of HIM-PROTACs were synthesized and evaluated, and two of them, GDCNF-2/GDCNF-11 potently induced ferroptosis via HSP90-mediated ubiquitin-proteasomal degradation of GPX4 in HT-1080 cells with DC50 values of 0.18 and 0.08 μM, respectively. In particular, GDCNF-11 showed 15-fold more ferroptosis selectivity over GPX4 inhibitor ML162. Moreover, these two degraders effectively suppress tumor growth in the mice model with relatively low toxicity as compared to the combination therapy of GPX4 and HSP90 inhibitors. In general, this study demonstrated the feasibility of degrading GPX4 via HSP90 interactome, and thus provided a significant complement to existing TPD strategies.

DOI: https://doi.org/10.1021/acs.jmedchem.4c01518
Nat Commun. 2024 Aug 29. 15(1): 7511

The novel ribosome biogenesis inhibitor usnic acid blocks nucleolar pre-60S maturation.

Lisa Kofler, Lorenz Grundmann, Magdalena Gerhalter, Michael Prattes, Juliane Merl-Pham, Gertrude Zisser, Irina Grishkovskaya, Victor-Valentin Hodirnau, Martin Vareka, Rolf Breinbauer, Stefanie M Hauck, David Haselbach, Helmut Bergler.

The formation of new ribosomes is tightly coordinated with cell growth and proliferation. In eukaryotes, the correct assembly of all ribosomal proteins and RNAs follows an intricate scheme of maturation and rearrangement steps across three cellular compartments: the nucleolus, nucleoplasm, and cytoplasm. We demonstrate that usnic acid, a lichen secondary metabolite, inhibits the maturation of the large ribosomal subunit in yeast. We combine biochemical characterization of pre-ribosomal particles with a quantitative single-particle cryo-EM approach to monitor changes in nucleolar particle populations upon drug treatment. Usnic acid rapidly blocks the transition from nucleolar state B to C of Nsa1-associated pre-ribosomes, depleting key maturation factors such as Dbp10 and hindering pre-rRNA processing. This primary nucleolar block rapidly rebounds on earlier stages of the pathway which highlights the regulatory linkages between different steps. In summary, we provide an in-depth characterization of the effect of usnic acid on ribosome biogenesis, which may have implications for its reported anti-cancer activities.

DOI: https://doi.org/10.1038/s41467-024-51754-3
Nat Chem Biol. 2024 Aug 30.

Engineering artificial non-coding RNAs for targeted protein degradation.

Congcong Cao, Aolin Li, Chaojie Xu, Baorui Wu, Lin Yao, Yuchen Liu.

Targeted protein degradation has become a notable drug development strategy, but its application has been limited by the dependence on protein-based chimeras with restricted genetic manipulation capabilities. The use of long non-coding RNAs (lncRNAs) has emerged as a viable alternative, offering interactions with cellular proteins to modulate pathways and enhance degradation capabilities. Here we introduce a strategy employing artificial lncRNAs (alncRNAs) for precise targeted protein degradation. By integrating RNA aptamers and sequences from the lncRNA HOTAIR, our alncRNAs specifically target and facilitate the ubiquitination and degradation of oncogenic transcription factors and tumor-related proteins, such as c-MYC, NF-κB, ETS-1, KRAS and EGFR. These alncRNAs show potential in reducing malignant phenotypes in cells, both in vitro and in vivo, offering advantages in efficiency, adaptability and versatility. This research enhances knowledge of lncRNA-driven protein degradation and presents an effective method for targeted therapies.

DOI: https://doi.org/10.1038/s41589-024-01719-w